A dose Dependent hepatoprotective and nephroprotective activity of eucalyptus oil on Streptozotocin induced diabetic mice model

Binomial name of the plant is Eucalyptus globulus Labill. These plants are commonly known as Tasmanian bluegum or southern bluegum. The plant name has been checked with http://​www.​theplantlist.​org. The accepted name record derives from WCSP(World checklist of selected plant families) data supplied on 2012–03-23. The original publication details are Voy.Rech.Perouse 1:153 1800.

Pure extracted oil was purchased from Auroshikha,Pondicherry and were used without further purification. To know the chemical constituents of the oil, they were chemically characterized by gas-chromatography. The condition for gas chromatography was mentioned as follows: Gas chromatography: The essential oils were analyzed using a Perkin-Elmer gas chromatograph model 8700, which is equipped with flame ionization detector (FID) and HP-5MS capillary column (30 m × 0.25 mm). Injector and detector temperatures were adjusted at 220–290 °C. Column oven temperature was set from 80 to 220 °C at the rate of 4 °C per minute. Initial and final temperatures were 3 and 10 min, respectively. Helium gas was used as carrier with flow of 1.5 mL per minute. A sample of 1.0 μL was injected, using slit mode (split ratio, 1:100). All quantification was done by a built-in data-handling program provided by the manufacturer of the gas chromatograph (Perkin-Elmer, Norwalk, CT, USA). The composition was reported as a relative percentage of the total peak area [8].

Adult albino mice of average weight between 29 and 30 g of both sexes were obtained and were acclimatized in the laboratory for 2 weeks under proper aeration and at a temperature of 22°celsius and normal atmospheric pressure and also under proper supervision. They had access to food and pure water ad libitum and the stress level was 0 at the time of experiment.

Diabetes was induced by single intraperitoneal injection of Streptozotocin (STZ) (Sigma Aldrich 75%, USA) (60 mg/kg body weight in 0.1 mol/L citrate buffer, pH 4.5) into 16–18 h fasted mice. 5% glucose solution was supplied to STZ-induced mice for the next 24 h to avoid hypoglycemic mortality. After 96 h of STZ injection, blood sample (10 μL) from mice which were induced with Streptozotocin was collected by tail snip method and glucose was monitored by Accu-Chek (Roche Accu-Chek Advantage® whole-blood glucose monitor).

Fasted mice with glucose concentration greater than 200 mg/dl (11.1 mmol/L) were considered as hyperglycemic [9].Therefore initial checking of all biochemical parameters without induction of hyperglycemia was consider as day 0.After 96 h (4^thday) only blood glucose was checked to confirm the induction of diabetes. Then at 7th, 14th, 21th, 28th day whole biochemical parameters were observed.

There were total 56 mice which were grouped into 7 sets containing 8 mice in each group.

Group I-Control (Normal diet). Group II to VII (STZ) and were diabetic. Group III (Glibenclamide [5 mg/kg] [10]. Group IV to VII (0.5%,1%,1.5%,2% Eucalyptus oil [EO]) respectively. EO was prepared by taking different percentage (0.5,1, 1.5, 2) of oil with respect to water as solvent and after immediate sonication which was done by ultrasonic sonicator stabilized the sample and immediately they were injected to the body .

EO were administered to mice at a dose of 0.5, 1, 1.5, 2, 2.5 (ml/kg body weight) and observe the behavioral change and mortality rate for a whole day.

A 28 days treatment was done and body weight and biochemical assays were analyzed. At the end of the treatment, mice from each group’s blood samples were collected from the retro orbital plexus then were sacrificed by anaesthetized. Blood serum was collected by repeated centrifugation at 3000×g for 10 min to assay blood parameters (Bilirubin, Cholesterol, SGPT, SGOT, ALP, GGT, Urea, Creatinine) For hepatoprotective enzymes like SGPT and SGOT tests modified UV (IFCC) kinetic assay was followed where absorbance was recorded at340nm.ALP was checked by p NPP –AMP (IFCC), kinetic assay. Absorbance was taken at 405 nm.GGT test was also done following the SZASZ method. ENZOPAK GGT is formulated on Szasz method recommended by Scandinavian Society for Clinical Chemistry and Physiology.This method is very particular in kinetic, specific and sensitive. Serum absorbance for GGT was taken at 405 nm.Bilirubin and cholesterol test followed Jendrassik and Grof method and CHOD-PAP method. Optical absorbance for Bilirubin was at 546 nm and 505 nm for cholesterol respectively. For nephroprotective enzymes like urea and creatinine Urease, Berthelot, end point assay and modified Jaffe’s reaction initial rate assay was followed. And optical absorption was 578 nm for urea and 505 nm for creatinine. Liver, kidney and pancreas were excised. For histology, excised liver, kidney and pancreas were washed in phosphate buffer and then transfer to 10% formalin fixative solution for 48 h. All three types of tissues were processed for paraffin embedding and were sectioned of 5 μm in microtome. After staining with haematoxylin and eosin, they were examined under microscope (Axio Scope A1-Zesis) at 100X magnification.

Data expressed as Mean ± standard deviation and all analysis was carried out by Microsoft office excel and Origin software (version Pro 8.5; Origin Lab Corporation, Northampton, MA 01060, USA.

Biochemical aspects of the blood of different group of mice serum depicted through different graphs.

In Fig. 2 a] study of body weight was done, group I showed no change in body weight but due to induction of Streptozotocin only (Group II diabetic) the body weight was decreased. But positive result was observed when mice were treated with 1.5% EO (Group VI). Conventional drug Glibenclamide was also help to maintain the normal body weight as expected. [12, 13]. The reason behind the loss of body weight after treated with STZ was STZ reduces the number and size of adipocytes. Also cells were unable to oxidize glucose and incorporate into lipid. Because of that, loss of adipose mass occurred and loss of body weight was seen 1.5% EO regain the normal weight by regaining the number of adipocyets [14] Figure 2 b,] blood glucose level of healthy mice with diabetic mice and diabetes recovered mice was observed. Normal blood glucose level was less than 200 mg/dL observed in group I but Streptozotocin damage the beta cell in pancrease through necrosis which further unable to produce insulin that leads to rise in glucose level in blood. [15]. EO of different doses especially 1.5% and Glibenclamide helped to normalize the whole system. [16, 17]. Glibenclamide class sulfonyl urea helps to increase the amount of insulin production. The mechanism of action of most of the common antidiabetic drug was seen that they bind to the K_ATP inhibitory regulator subunit named sulfonyl urea receptor 1 in pancreatic beta cell. Inhibition cause cell membrane depolarization and result in opening voltage gated calcium channel and increase the amount of intracellular calcium (ca2+) in beta cell which cause stimulation of insulin release. Post effect of EO treatment has been shown a dramatic reduction of high glucose which confirms the high level of insulin which further promotes an idea that this insulin are secreted only because of the stimulation of pancreatic beta cells. So probability of mechanism of EO as drug may follow the same pathway like other conventional drug which requires further extended analysis.

In Fig. 2 c] Normal Bilirubin range was observed in control group but higher in STZ induced group. Bilirubin is the main bile pigment which formed because of breakdown of heme of red blood cells by reticuloendothelial system. Unconjugated bilirubin is carried by Albumin to the liver where it is conjugated with glucuronic acid and becomes water soluble. Total Bilirubin concentration always increases severely in hepatocellular disease. Here also increased Bilirubin was observed as STZ caused hepatocellular damage but after EO treatment again normal range of Bilirubin was seen, especially when treated with 1.5%.EO. [18, 19, 20]. Figure 2 d , showed the distinct variation of total cholesterol among normal, diabetes and diabetes recovered mice. Control group have normal range of cholesterol, followed by increased in cholesterol range in group II. Cholesterol is a waxy substance made by the body and found in some animal-based foods. Blood cholesterol levels describe a group of fats also known as lipoproteins that include HDL and LDL. Cholesterol can be harmful by contributing to narrowed or blocked arteries. Result showed that conventional drug Glibenclamide drags the level of cholesterol into normal range. Like the same EO of 1.5% also depicted the same result. As 261 mg/d L is quite high at 7th day but it reduces drastically to 122 mg/d L on 28th day [21].

SGPT and SGOT are elevated in viral and other liver diseases associated with Hepatic necrosis. SGPT is more liver specific enzyme. Medium increase of these enzymes may be seen in Cirrhosis, Extrahepatic cholestasis, and carcinoma of liver. When the liver cells get damaged or injured, these enzymes came into the blood stream, raising their percentage in blood levels. Hence raised blood levels of SGOT and SGPT signifies liver disease or injury. As result observed in Fig. 3 a and b where STZ induced blood serum had high SGPT & SGOT level confirming liver damage which can be rectified by EO 1.5% treatment [18, 19, 22]. Figure 3 c depicted the comparative analysis of serum level of Alkaline phosphatase (ALP) of different treated group of mice. Clinical significance of ALP showed detecting liver related diseases or bone disorders. When liver was affected by STZ, cells released increase amount of ALP into the blood stream. This test is also used to detect blocked bile ducts because ALP is high especially in the edge of cells that join to form bile duct. Elevated ALP result could confused whether they got elevated because of live or bone disorder. So for further confirmation GGT may also be done because GGT level always increased in liver diseases but not in bone disorders. Elevated ALP like 113 IU/DL had been observed in serum of STZ induced mice, which can be reduced and came to normal level when EO of different dose was used as drug especially when 1.5% EO was applied. ALP is usually much less elevated than SGPT and SGOT when bile ducts are blocked (usually gallstone, scars from surgery or by cancer. [18, 19, 21, 23]. GGT functions in the body as a transport molecule. It helps to move other molecules around the body which has a significant role in helping the liver to metabolize drugs and other toxins.GGT is concentrated in the liver, but it’s also present in the gallbladder, spleen, pancreas, and kidneys. GGT blood levels are usually high when the liver is damaged. This test is often done with other tests that measure liver enzymes if there’s a possibility of liver damage [24]. In this Fig. 3 d, group II has possibilities of liver damage as confirm by elevated GGT result whereas significant results could be seen in group VI where treated drug EO helped in lowering GGT to normal level [25].

Figures 1, 4 a and b are the data depicted urea and creatinine level of serum of different groups of treated mice. Both elevated urea and creatinine of the serum treated with STZ can be observed in both the figures and normalized range of these kidney enzymes were acquired by treating the model with 1.5% EO. Patient with chronic liver disease have a significantly lower baseline serum creatinine concentration than the general population [26, 27]. Urea and creatinine are the two enzymes generally used in testing nephroactivity as they are the chief indicator of kidney function. Abnormally high levels of creatinine give a warning of possible malfunction or failure of the kidney. Urea (BUN) is another indicator of kidney function.BUN to creatinine ratio generally provides more precise information about kidney function.

Survival rate was 100% and no reports of death was seen even after 28 days.

The above figure showed the histopathological study of liver tissue which depicted the change of cell morphology before and after the treatment of eucalyptus oil(EO). In Fig. 5 a we can see the normal structure of central vein (marked) also the hepatocyte and sinusoids are well preserved and demarcated.. Cytoplasm is not vacuolated. Coming to Fig. 5 b cell morphology was not normal and having abnormal structure of central vein, vacuolated cytoplasm was observed, many hepatocytic cells were found to be necrotized near central vein. Figure 5 c tissues were treated with Glibenclamide and regeneration of hepatocytic cells were noticed followed by regaining the normal shape of central vein and the shape of the normal cell was regained. Figure 5 [d, e, f, g] depicted the effect of EO. And the best result showed by Fig. 5 f where 1.5%EO was applied where we can see complete structure of central vein and hepatocyte with normally visible sinusoid and less vacuolated cytoplasm was observed. Necrosis, congestion of the blood vessel and inflammatory infiltration was less observed. Overall analysis exhibited that 1.5% EO helps to restore the normal architect of liver tissue. Prominent central vein structure and hepatocytes were marked in the Fig. 5 (d, e, f, and g) [4, 28, 29, 30].

Figure 6 a showed that is control without diabetes showed the normal architect glomerulus surrounded by Bowman’s capsule. Distal convoluted tubules and proximal tubules were observed. In Fig. 6 b the tissues were treated only with STZ and the structural abnormality can easily be identified having abnormal glomerulus with infiltration in proximal tubule. In Fig. 6 c the kidney tissues were healed with the help of Glibenclamide. In case of Fig. 6 d where tissues were treated with 0.5% EO, in some place shrinkage of glomeruli and tubular infiltration was noticed. But as the dose of the drug EO changed from 0.5 to 1%(Fig. 6 e) pathological percentage of damage in structure of kidney section reduced significantly and the best result was depicted by the tissue when treated with 1.5% EO (Fig. 6 f) where no messengial expansion or messengial cell proliferation or thickening of glomerulus basement membrane was observed. Regeneration of new healthy tissue was shown with normal architect. But there is no significant improvement showed by tissue treated with 2% EO (Fig. 6 g) [4, 31].

Here in Fig. 7 a showed normal tissue architecture where islet of langerhans was prominently visible with acinar cells were arranged in lobules (marked with arrows).

But in case of STZ treated tissues (Fig. 7 b), the structural morphology was not normal; size of the islet cell was shrunken with architectural disarray (marked) and number of acinar cells decrease around the islet cell. When the tissue was treated with Glibenclamide then in usual manner the tissue got recovered and normal morphology was gained gradually. When EO was treated, 0.5% didn’t show any significant change but 1% showed proper structure of islet cells (Fig. 7 e) with increased number of acinar cells. But best result was obtained when 1.5% was treated, at that time better restoration of the beta cell in comparison to other dose was observed. Normal size of islet cells with normal number of acinar cells with less necrotic cell and contracted blood vessels were observed (Fig. 7 f). When 2% was applied there was improvement in the number of acinar cells but no improvement was took place for islet of langerhans cells [4, 32]. As the dose of Streptozotocin was 60 mg/kg body weight which has been consider as a moderate dose for the induction of diabetes and it didn’t damaged all of the pancreatic cells which was proved by the Histopathological image of pancrease as shown in Fig. 7 and also proved that 1.5% dose of Eucalyptus oil has the capability to regenerate the damaged pancreatic cells. As this work is a basic preliminary work which emphasized only the dose dependant activity of the eucalyptus oil on diabetes, so insulin release was not considered as a checking parameter.